Bottom Line:
The site for CNTFRα enables CLF-1 to promote CLC:CNTFRα complex formation and signaling.The second site establishes a link between the endocytic receptor sorLA and the tripartite CLC:CLF-1:CNTFRα complex and allows sorLA to downregulate the CNTFRα pool in stimulated cells.Finally, sorLA may bind and concentrate the tripartite soluble CLC:CLF-1:CNTFRα complex on cell membranes and thus facilitate its signaling through gp130/LIFRβ.

Figure 1: SPR analysis of the binding of CLC:CLF-1, CLC, and CLF-1 to immobilized sCNTFRα and to the immobilized luminal domain of sorLA, as indicated. (A) CLC:CLF-1 binding (at 100 nM) to sCNTFRα. (B) Binding of the individual subunits CLC and CLF-1 to sCNTFRα. Ligand concentrations and estimated Kds are indicated, and an SDS-PAGE analysis of the purified CLF-1 is shown in the inset (Coomassie stain). (C) Concentration dependence of CLC:CLF-1 binding to sorLA. The dotted line indicates CNTF binding. (D) Inhibition of CLC:CLF-1 binding to sorLA following preincubation of sorLA with sorLA-propeptide. Immobilized sorLA was first exposed to either unsupplemented binding buffer (dotted line) or to buffer containing sorLA propeptide (5 μM; solid line), and CLC:CLF-1 was subsequently added as indicated (arrows). (E) Concentration dependence (and Kd) of CLF-1 binding to sorLA. The dotted line indicates binding of CLC (250 nM) to sorLA. (F) Inhibition of CLF-1 binding to sorLA preincubated with sorLA-propeptide. sorLA was subjected to unsupplemented buffer (dotted line) or to buffer containing a high concentration (5 μM) of sorLA propeptide (solid line) prior to addition of CLF-1 as indicated. (G and H) Induction of pSTAT3 in SH-SY5Y cells. The cells were starved for 4 h prior to stimulation (15 min) with 10 nM CLC, CLF-1, CLC:CLF-1, or CNTF (as a positive control) and subsequently lysed in the presence of phosphatase inhibitors. Induction of pSTAT3 was determined by reducing SDS-PAGE and Western blotting using anti-pSTAT3 antibodies. Panel G shows detected pSTAT3 and corresponding levels of β-actin of a single experiment. Results of four similar experiments (means ± SEM) are shown in panel H.

Mentions:
We have previously reported that the Vps10p-D receptor sortilin binds the two CNTFRα ligands CNTF and CLC:CLF-1 (20). Since sortilin and sorLA are members of the same receptor family and are known to share several ligands, we initially examined whether sorLA also exhibited affinity for the two ligands. We found that sorLA, unlike sortilin, did not bind to CNTF, whereas the heterodimeric CLC:CLF-1 bound to sCNTFRα as well as to the ectodomain of sorLA (Fig. 1A and C). Analysis by SPR demonstrated that binding to immobilized soluble sorLA, i.e., the luminal part of full-length sorLA, exhibited high affinity (Kd of 5 ± 2.9 nM; n = 3), as determined by binding at different concentrations of CLC:CLF-1 (Fig. 1C). The binding was completely abolished in the presence of a surplus of sorLA's own propeptide, demonstrating that CLC:CLF-1 selectively targets the Vps10p-D β-propeller (Fig. 1D). Binding was also efficiently inhibited by receptor-associated protein (RAP) but almost unchanged by neurotensin (NT) (data not shown).

Figure 1: SPR analysis of the binding of CLC:CLF-1, CLC, and CLF-1 to immobilized sCNTFRα and to the immobilized luminal domain of sorLA, as indicated. (A) CLC:CLF-1 binding (at 100 nM) to sCNTFRα. (B) Binding of the individual subunits CLC and CLF-1 to sCNTFRα. Ligand concentrations and estimated Kds are indicated, and an SDS-PAGE analysis of the purified CLF-1 is shown in the inset (Coomassie stain). (C) Concentration dependence of CLC:CLF-1 binding to sorLA. The dotted line indicates CNTF binding. (D) Inhibition of CLC:CLF-1 binding to sorLA following preincubation of sorLA with sorLA-propeptide. Immobilized sorLA was first exposed to either unsupplemented binding buffer (dotted line) or to buffer containing sorLA propeptide (5 μM; solid line), and CLC:CLF-1 was subsequently added as indicated (arrows). (E) Concentration dependence (and Kd) of CLF-1 binding to sorLA. The dotted line indicates binding of CLC (250 nM) to sorLA. (F) Inhibition of CLF-1 binding to sorLA preincubated with sorLA-propeptide. sorLA was subjected to unsupplemented buffer (dotted line) or to buffer containing a high concentration (5 μM) of sorLA propeptide (solid line) prior to addition of CLF-1 as indicated. (G and H) Induction of pSTAT3 in SH-SY5Y cells. The cells were starved for 4 h prior to stimulation (15 min) with 10 nM CLC, CLF-1, CLC:CLF-1, or CNTF (as a positive control) and subsequently lysed in the presence of phosphatase inhibitors. Induction of pSTAT3 was determined by reducing SDS-PAGE and Western blotting using anti-pSTAT3 antibodies. Panel G shows detected pSTAT3 and corresponding levels of β-actin of a single experiment. Results of four similar experiments (means ± SEM) are shown in panel H.

Mentions:
We have previously reported that the Vps10p-D receptor sortilin binds the two CNTFRα ligands CNTF and CLC:CLF-1 (20). Since sortilin and sorLA are members of the same receptor family and are known to share several ligands, we initially examined whether sorLA also exhibited affinity for the two ligands. We found that sorLA, unlike sortilin, did not bind to CNTF, whereas the heterodimeric CLC:CLF-1 bound to sCNTFRα as well as to the ectodomain of sorLA (Fig. 1A and C). Analysis by SPR demonstrated that binding to immobilized soluble sorLA, i.e., the luminal part of full-length sorLA, exhibited high affinity (Kd of 5 ± 2.9 nM; n = 3), as determined by binding at different concentrations of CLC:CLF-1 (Fig. 1C). The binding was completely abolished in the presence of a surplus of sorLA's own propeptide, demonstrating that CLC:CLF-1 selectively targets the Vps10p-D β-propeller (Fig. 1D). Binding was also efficiently inhibited by receptor-associated protein (RAP) but almost unchanged by neurotensin (NT) (data not shown).

Bottom Line:
The site for CNTFRα enables CLF-1 to promote CLC:CNTFRα complex formation and signaling.The second site establishes a link between the endocytic receptor sorLA and the tripartite CLC:CLF-1:CNTFRα complex and allows sorLA to downregulate the CNTFRα pool in stimulated cells.Finally, sorLA may bind and concentrate the tripartite soluble CLC:CLF-1:CNTFRα complex on cell membranes and thus facilitate its signaling through gp130/LIFRβ.